Carole Bewley LBC, NIDDK, NIH Annual Report, Fiscal Year 2001 The Natural Products Group of LBC, NIDDK, is active in two areas of research, both of which seek to identify and understand the structural basis and mechanisms of action of inhibitors to (1.) mycothiol-dependent biosynthesis and detoxification, and (2.) HIV fusion. 1. In eukaryotes, glutathione is the primary thiol involved in maintaining a reducing intercellular environment and in protecting cells from both oxidative stress and alkylating agents. In contrast, many prokaryotes (especially Gram-positive bacteria) employ alternative small molecular weight thiols that are unique to particular bacterial groups. In the case of actinomycetes, which include the mycobacteria, myo-D-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-a-D-glucopyranoside (mycothiol, MSH),1 , is the only thiol present and is thought to function similarly to glutathione in eukaryotes. Two key enzymes from Mycobacterium tubercuolsis (strain Rv37) that are involved in either the biosynthesis of mycothiol (rv1170 coding for AcCys-Ins deacetylase), or in mycothiol-dependent detoxification (rv1082 coding for mycothiol-S-conjugate amidase, hereafter referred to as MCA), have been identified recently. Given the unique structure and distribution of mycothiol, along with the absence of detectable homology between rv1170 or rv1082 and other gene sequences available in public databases, these enzymes represent worthy targets for new classes of Gram-positive-specific antibiotics or antituberculars. Thus, one aspect of our research aims to identify novel small-molecular weight inhibitors to the M. tuberculosis. deacetylase and amidase enzymes, and ultimately to determine the structural basis for the biosynthesis of mycothiol and inhibition of MCA. To this end we have thus far screened ~ 1500 organic extracts from diverse collections of marine plants, invertebrates, and terrestrial fungi (NCI Natural Products Repository) for their ability to inhibit MCA. Our initial goal in selecting this library was to obtain a profile of chemical structures that have the ability to inhibit MCA. Using a flouroescence-based HPLC assay that detects the presence of a fluorescent bimane derivitive of MSHmb and its MCA cleavage product AcCys-mb, we have identified a number of novel and known natural products that inhibit MCA at submicromolar to micromolar concentrations, at least one of which has been reported to be lethal to M. tuberculosis. Although the genes encoded by rv1170 and rv1082 exhibit ~30% homology, the substrate preferences are significantly different. We are currently investigating the degree of inhibition of the MCA inhibitors on the deacetylase, as well as screening for inhibitors of the deacetylase. Ongoing synthetic work includes the synthesis of a putative precursor to MSH, namely GlcNAc-a(1?1)myo-D-inositol; of mycothiol; and eventually of analogs of each that will be used to study substrate specificity. Mindful of both the chemical structures of the inhibitors isolated thus far and the structures of the natural substrates, future studies will include the design and synthesis of substrate-based inhibitors. 2. In order for enveloped viruses to infect cells, membrane fusion must occur. In the case of HIV, the events leading to fusion include binding of the HIV envelope glycoprotein gp120 to the primary receptor CD4, and subsequent binding to the chemokine receptors CXCR4 or CCR5, which together facilitate insertion of the fusion peptide of gp41 (i.e. the transmembrane subunit of the HIV envelope protein) into the host cell membrane, ultimately leading to fusion. Current anti-retrovirals target exclusively HIV protease and reverse transcriptase (RT). While we have witnessed remarkable success in the design and development of protease and RT inhibitors, these drugs are often poorly tolerated and prohibitively expensive for much of the underdeveloped world. The second aspect of our research focuses on inhibitors of viral entry, and encompasses the following projects: (i.) identification of small-molecule inhibitors to HIV fusion, (ii.) determination of the structural basis of the potent fusion-blocking activity of cyanovirin-N, and (iii.) the design of proteinaceous inhibitors and/or antigens of HIV fusion (not addressed below, see Ref. 7). i.) To date potent inhibitors to viral entry by HIV are limited to peptides or proteins comprising sequences from HIV-Env, to chimeric proteins incorporating neutralizing antibodies, or to compounds of modest molecular weight (1-5 kDa) that bear an overall abundance of negatively charged moeities. In the 3rd example, binding to Env is thought to be nonspecific and to occur through interactions with the positively charged coreceptor-binding regions on gp120. Thus, the impetus for attempting to identify small-molecule inhibitors to HIV fusion stems not only from the desire to identify new classes of potential antivirals, but also to reveal novel scaffolds that mimic naturally occurring proteins. As in Part 1 above, we have thus far screened ~1500 extracts in a quantitative vaccinia virus-based fusion assay (Berger and coworkers) that faithfully reproduces the events leading to HIV-1 Envelope-mediated fusion , and have identified a number of new and known natural products, several of which inihibit fusion at submicromolar concentrations. These include novel bishomoscalarane sesquiterpenes and guanidinium alkaloids. ii.) Cyanovirin-N (CVN) is an 11kDa cyanobacterial protein that potently inhibits (IC50?1nM) all strains of HIV and SIV at the level of fusion via high affinity carbohydrate-mediated interactions with gp120. Through a series of competition experiments using the fusion assay and a panel of complex and oligomannose-type oligosaccharides known to be present on fusogenic gp120 expressed in mammalian cells, we have recently shown that CVN selectively binds to the D1D3 isomer or oligomannose-8 (Man8 D1D3) and to oligomannose-9 (Man9) with nanomolar affinity, that these carbohydrates act as divalent and trivalent ligands to CVN at nanomolar concentrations, and that they directly compete with fusogenic HIV-1 Env to fully inhibit the fusion-blocking activity of CVN. Owing to the structures of Man8 D1D3 and Man9 relative to other high-mannose oligosaccharides (which cannot compete with gp120 for CVN binding), we have shown that Mana(1-2)Mana, which represents the terminal disaccharide of the full-length D1 and D3 arms of oligomannose, is also a high affinity ligand for CVN. Using NMR we followed the 1H-15N correlation spectrum of CVN titrated with Mana(1-2)Mana to unambiguously demonstrate that CVN contains two novel carbohydrate binding sites of differing affinities located at opposite ends of the pseudo-symmetrical protein. We have also solved the solution structure of a 1:2 CVN:Mana(1-2)Mana complex using multi-dimensional heteronuclear NMR to reveal the first structure of a complex of a mannose-specific carbohydrate-binding protein with nanomolar affinity. The presence and location of two opposing carbohydrate binding sites in conjunction with the geometrical restraints imposed by the spacing of the D1 and D3 arms of oligomannose presents a simple model for CVN-gp120 binding.